The efficacy of intrauterine infusion of platelet rich plasma in women undergoing assisted reproduction: a systematic review and meta-analysis

Background Platelet-rich plasma (PRP) is an autologous platelet concentration recently used in the reproductive field. Studies had conflicting results regarding its effect on pregnancy outcomes. We aimed to solve the debate on the safety and efficacy of PRP in women undergoing assisted reproduction and assess the influence of covariates on the outcomes of PRP infusion. Methods We searched PubMed, Scopus, Cochrane, and Web of Science in May 2023. We included randomized and non-randomized clinical trials as well as cohort studies assessing intrauterine PRP in sub fertile women undergoing assisted reproduction (IVF/ICSI). For the quality assessment, We used the Cochrane Risk of Bias Tool 1, the ROBINS-I tool, and the Newcastle–Ottawa Scale. We pooled the data using RevMan version 5.4. Results The data from 23 studies were pooled. PRP had favorable outcomes compared with the control group on clinical pregnancy rate (RR: 1.84, 95% CI 1.62 to 2.09; P < 0.00001), live birth rate (RR: 1.75, 95% CI: 1.24 to 2.47; P = 0.001), and miscarriages (RR: 0.51, 95% CI: 0.36 to 0.72; P = 0.0002). Women with repeated implantation failure had a significantly improved clinical pregnancy rate (RR: 1.83, 95% CI: 1.49 to 2.24; P < 0.00001), live birth rate (RR:1.83, 95% CI: 1.33 to 2.51; P = 0.002), and miscarriage rate (RR: 0.46, 95% CI: 0.31 to 068; P = 0.0001). Conclusion PRP showed promising results in assisted reproductive techniques. Further large and multicenter RCTs are required to compare the doses of PRP while identifying the specific population with the most benefits from PRP. Supplementary Information The online version contains supplementary material available at 10.1186/s12884-023-06140-0.


Background
Although assisted reproduction techniques significantly improved conception rates, the issue of implantation failures remains unsolved.This can be attributed mostly to poor endometrial receptivity and embryo endometrial communication where achieving an implantation necessitates a receptive endometrium, a functional embryo, and a coordinated communication between them [1].This happens naturally five to seven days after ovulation.The endometrial receptivity then is optimum for embryo implantation [2].Endometrial receptivity can be affected by many factors including anatomical uterine abnormalities and endometrial thickness among others [3].Thin endometrium of less than 7 mm is frequently linked to poor conception outcomes such as recurrent implantation failure (RIF) [4].RIF is defined as the implantation failure of at least three successive in vitro fertilization (IVF) treatments with good quality embryos [5].It constitutes a major economic and psychologic problem [6].Thus, it is essential to find an effective treatment that can improve pregnancy outcomes.
Currently, there is no consensus on the optimal approach.In some women, the hormonal therapy might be unsuccessful in increasing the thickness of the endometrium [7].Moreover, irrespective of endometrial morphometry, the endometrial blood flow was impaired during follicular phase in patients with unexplained implantation failures [8].New therapeutic options have been suggested to enhance pregnancy rates for women with implantation failures.These interventions include intra-uterine granulocyte colony stimulating factor (G-CSF), intra-uterine human chorionic gonadotropins, and intra-uterine platelet rich plasma (PRP).The network meta-analysis by Jin et al. revealed that among these interventions, PRP was the most effective among women with 2 or more implantation failures [6].
Platelets are small non-nucleated cellular fragments involved in homeostasis derived from megakaryocytes with a short life span [9].They have granules that store various cytokines, and growth factors.At the site of inflammation or injury, platelets are activated and several factors are released including fibroblast growth factor, platelet-derived growth factor, tumor growth factor-β, and vascular endothelial growth factor.[10].Therefore, administering a platelet concentrate involves infusing a huge quantity of cytokines and chemokines that enhance immunity, healing, and regeneration.[10].PRP is an autologous platelet concentration in plasma.For the preparation of PRP, blood is drawn from a peripheral vein, kept in the anticoagulant citrate dextrose solution then processed to enhance platelets by separating distinct components of blood [11].It has recently been identified as an effective therapy in many fields.
The role of PRP in sub fertile women was first investigated by Chang et al. [12].They found that PRP improved endometrial thickness and pregnancy outcomes.Therefore, several studies investigated the efficacy of PRP.However, they came with conflicting results.Some studies [13,14] found no difference in the risk of miscarriages while Nazari et al. [15] showed that PRP had significantly reduced miscarriages.Some studies [16][17][18] demonstrated that PRP infusion had insignificant effect on clinical pregnancy rate while others [15,19,20] showed that PRP improved it significantly.Since the studies had inconsistent results, we conducted our systematic review and meta-analysis to investigate the role of intrauterine infusion of PRP on conception outcomes, and solve the ongoing debate.We also aimed to assess the effect of covariates on the outcomes of PRP infusion.

Methods
We conducted our systematic review and meta-analysis following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.[21].We followed the Cochrane handbook guidelines in doing all the steps [22].

Search strategy
We searched Cochrane, PubMed, Web of Science, and Scopus in June 2022 then updated it in May 2023.We used the following keywords in our search strategy ("platelet rich plasma" OR "platelet gel" OR PRP) AND ("in vitro fertilization" OR "embryo transfer" OR RIF OR "embryo implantation").The supplementary file contains the search strategy.We searched clinicalTrials.govmanually, and protocols without published results in a peer reviewed journal were excluded.

Study selection
Two authors in two steps manually screened the retrieved studies.At first we screened the studies according to their title and abstract then we screened the full-text of eligible studies.For any discrepancies, a third author was consulted.We included randomized clinical trials (RCTs), cohort studies, and non-randomized controlled trials comparing intrauterine infusion of platelet-rich plasma with no PRP or placebo in sub fertile women undergoing assisted reproduction (IVF/ICSI).
Abstracts, reviews, editorials, single arm trials, case series, and non-English studies were excluded.

Quality assessment
For assessment of the included studies, two authors independently evaluated them.For RCTs, we used the Cochrane risk of bias tool 1 (ROB1) [23].The judgement of the authors is classified as low risk, unclear risk, or high risk of bias.If there was a disagreement, a third author was consulted.We used the ROBINS-I tool [24] for evaluating the quality of non-randomized studies.We used the Newcastle-Ottawa Scale (NOS) [25] for cohort studies.

Data extraction and study outcomes
The authors performed the data extraction in prepared formatted excel sheets.The characteristics of the studies included: inclusion and exclusion criteria, study ID, center (country), intervention and control arms, study design, and reported outcomes.
The baseline data included the age, etiology and duration of infertility, body mass index, type of infertility, number of embryos transferred, previous implantation failure, and endometrial thickness.
The primary outcomes were clinical pregnancy, live birth, and miscarriages.
The secondary outcomes were implantation rate, chemical pregnancy, endometrial thickness, ectopic pregnancies, multiple pregnancies, and ongoing pregnancies.Methods indicated in the Cochrane manual were used to deal with any incomplete or incompatible data.[22].

Statistical analysis
For endometrial thickness, mean difference (MD) and its 95% confidence interval (CI) were calculated, while the risk ratios (RR) with 95% CI were calculated for dichotomous variables.We used a fixed-effect model if there is no heterogeneity (P > 0.05); otherwise, a random-effect model was used.We assessed the statistical heterogeneity using the I 2 statistic where p-value of less than 0.05, I 2 more than 60% indicated heterogeneity .We conducted sensitivity analysis through exclusion of the study with the highest heterogeneity.We calculated miscarriages, multiple pregnancy, and ectopic pregnancies per the number of clinical pregnancies.Implantation rate was determined as the number of gestational sacs per the overall number of transferred embryos.
We performed the meta-analysis using Revman software 5.4.For the assessment of publication bias, we visually inspected the symmetry of funnel plot.We performed subgroup analysis for women with thin endometrium less than (7 mm), and RIF with 3 or more implantation failures.We performed a meta-regression using open meta-analyst to investigate the influence of age, BMI, duration of infertility, endometrial thickness, number of previous cycles, and number of embryos transferred on clinical pregnancy, chemical pregnancy, and miscarriages.

Study characteristics
Table 1 demonstrates a summary of the characteristics of the included studies.Studies were carried out between 2014 and 2021, of which 13 studies took place in Iran, four studies in China and a single study from every country of these (Bahrain, Egypt, India, Russia, Turkey, Saudi Arabia, and USA).We have included 14 RCTs [15-17, 20, 29-38], three non-RCTs [18,26,28] and six cohort studies [13,14,19,27,37,40].

Quality assessment
The included RCTs were appraised using the Cochrane ROB 1.As for the random sequence generation domain, all the studies were considered low risk except for one study [17] that was at high risk and two studies [33,39] whose risk was unclear.Regarding the allocation concealment domain, there was inadequate information in most of the studies to permit judgment of low or high risk.However, three studies [20,35,38] were considered to be of low risk, and one [17] was judged as high risk.
All studies were at low risk for performance and detection biases.
Regarding missing data, four trials were a source of a high risk of attrition bias, two of them [15,32] had a significant percentage of loss to follow-up and the other two [20,39] had unequal distribution to the loss of follow up, the remaining trials had a low risk of attrition bias.
Regarding reporting bias, all studies had low risk except for three trials [17,31,32] that did not report one of the primary outcomes and one study that did not provide sufficient information to judge [39].
Most studies were free from any other source of bias, except for five studies, which carried a high risk of bias.Pourkaveh et al. [39] had small study size.Allahveisi et al. [17], had small study size and vagueness regarding the causes of RIF.In Rageh et al. [33], the provided NCT was not found.Safdarian et al. [34] reported that the live birth rate was more than clinical pregnancy rate while Zargar et al. [35] had a wide range of age in its participants.
The summary of the quality assessment for the included 14 RCTs is shown in Figs. 2 and 3.
Regarding the assessment of the three included nonrandomized trials, and depending on the three domains of ROBINS-I, Abou-El-Naga et al. [26] were judged to have low risk, but both Dzhincharadze et al. [28] and Tehraninejad et al. [18] were judged to carry moderate to high risk of bias.See the Supplementary Table 1 for the details regarding the scoring of the non-RCTs.
Regarding the three domains of NOS (Selection, Comparability and Outcome), all included studies had good quality except for two studies had fair quality [38,39].See Supplementary Table 2 for the details regarding the scoring of the cohort studies according to NOS.
To investigate publication bias, we conducted funnel plots.Supplementary Figures 1 and 2 display the funnel plots for the clinical pregnancy rate and the chemical pregnancy rate in the entire population, respectively, the funnel plot exhibits asymmetry at the bottom, suggesting that studies with unfavorable results and smaller sample sizes were underrepresented, potentially indicating publication bias.For the miscarriage rate, in Supplementary Fig. 3, the funnel plot displays an asymmetrical appearance, indicating the possibility of publication bias.

Meta regression
For all the population, there was no significant relation between age, BMI, duration of infertility, endometrial thickness, number of previous cycles, and number of embryos transferred on clinical pregnancy, chemical pregnancy, and miscarriages.
For women with RIF, regarding chemical pregnancy, no significant relation was found with age, BMI, and duration of infertility.Regarding clinical pregnancy, we found no significant relation with age, BMI, and number of previous cycles.There was a significant relation between duration of infertility and clinical pregnancy rate (95% CI: 0.0 to 0.047; P = 0.049, Supplementary Fig. 33).
For women with implantation failures, regarding chemical pregnancy, we found no significant relation with age, BMI, and duration of infertility.Regarding clinical pregnancy, there was no significant relation with age, BMI, and duration of infertility.There was a significant relation between number of previous cycles and clinical pregnancy rate (95% CI: 0.019 to 0.584; P = 0.037, Supplementary Fig. 34).

Discussion
We investigated the role of intrauterine PRP among sub fertile women undergoing assisted reproduction and the effect of covariates on the outcomes of PRP.Our systematic review included 23 studies with 2,449 patients.There were 1,229 women receiving intrauterine platelet rich plasma and 1,220 women in the control group.Our analysis on all the included women revealed that PRP significantly improved clinical pregnancy, live birth, miscarriages, implantation rate, chemical pregnancy, ongoing pregnancy, and endometrial thickness whereas insignificant on multiple pregnancy, ectopic pregnancy.As for clinical pregnancy, the same findings were found among the analysis of RCTs, RIF, and thin endometrium.For RIF patients, PRP significantly improved live birth but had no significant effect in the analysis of RCTs.There was no statistically significant effect on miscarriages in the analysis of RCTs, whereas significant among women with RIF and those with thin endometrium.We found a statistically significant relation between clinical pregnancy and the duration of infertility among women with RIF, and with the number of previous cycles among women with implantation failure.
The role of PRP was first investigated in the meta-analysis conducted by Maleki-Hajiagha et al. [41].However, since the meta-analysis was the first to be conducted, some limitations were considered as they didn't investigate the role of PRP in live birth, their analysis was based on only 7 studies with 625 women.In one them [42], the control group were on systemic G-CSF.In contrast, among our included studies, systemic G-CSF was administered in both groups in Nourshin et al. [13].Among the overall population, our results were consistent with them regarding chemical pregnancy, clinical pregnancy, and implantation rates, and endometrial thickness but with larger sample size.However, our results came conflicting regarding miscarriages.
Several meta-analyses were carried out afterwards to investigate the role of PRP in the reproductive field.However, each study had some limitations.Liu et al. [43] combined the effect of invasive sub-endometrial and non-invasive intrauterine infusion.li et al. [44] didn't follow a strict definition for RIF.Maged et-al.[45] included self-controlled trials.The results of the meta-analyses showed that PRP improved clinical pregnancy [43][44][45][46][47], however they had inconsistent results regarding the risk of miscarriages.Regarding RIF patients, our results were conflicting with the meta-analyses conducted by Anitua et al., li et al., and were consistent with deng et al., and liu et al. [43,44,46,47].
The conflict among the previous meta-analyses on the risk of miscarriages among RIF patients can be attributed to many factors including the criteria for defining RIF, and the number of included studies.The inconsistency in results among the studies as concluded by Noushin et al. is attributable to the absence of consensus on the ideal method for preparation of PRP.Most of the studies did not mention the platelet or white blood cells quantification used in the PRP which would highly influence the results [13].
The role of PRP in improving pregnancy outcomes was believed to be related to its effect on endometrial thickness ever since the study conducted by Chang et al. [12], as there was an association between them.However, this is still questionable.Kim et al. [48] found that although PRP had favorable effect on pregnancy outcomes, no association was found between them.Moreover, it has been suggested that endometrial thickness is a poor predictor of clinical pregnancy [1].In our study, PRP significantly improved clinical pregnancy and endometrial thickness.However, in the meta-regression, we found no significant relation between them.
The precise mechanism behind PRP's positive impact is still unknown.However, it is suggested that this effect is due to its immunological role where providing an antiinflammatory endometrial environment hinders the rejection of implantation [49,50].This is done through the regulation of several inflammatory cytokines including interleukin1, interleukin 8, and interleukin 1-β [41].
Our strengths is that our review is comprehensive with large sample size.We followed PRISMA guidelines.All our included RCTs were considered low risk in performance bias and detection bias.We investigated the role of PRP on the risk of ectopic pregnancy.This outcome wasn't investigated in the previous meta-analyses.Our included studies come from 9 different countries across different continents, so our results could be generalizable.We didn't combine the effect of different methods of PRP administration in contrast to the meta-analysis by Liu et al.We followed a strict definition for RIF patients.
Our limitations is that we only considered studies in English.Most of our included RCTs had unclear allocation concealment.Publication bias was observed among the included studies.There was heterogeneity in the analysis as there was heterogeneity among the studies in the methods of preparation of PRP and subsequently the heterogeneity in the concentrations of platelets used for each dose of therapy.Where the same dose of PRP had different concentrations of platelets across different studies.
A standardized protocol is needed for the preparation of PRP in order to investigate the optimum dose for therapy.We recommend that further RCTs should investigate the optimum dose of PRP, and the role of PRP for different causes cause of subfertility.

Conclusion
PRP improved clinical pregnancy, live birth, and miscarriage rates in women undergoing IVF/ICSI.Further RCTs are needed to investigate optimum dose of PRP.

Fig. 1
Fig. 1 Shows the PRISMA flow chart, which summarizes the literature search, screening, and the number of included studies

Fig. 2
Fig. 2 Risk of bias summary.It shows a summary of the risk of bias for each included study

Fig. 3 Fig. 4
Fig. 3 Risk of bias graph for included studies

Fig. 5 Fig. 6
Fig. 5 Forrest plot for the effect of PRP-therapy on live birth rate after leave one out.(CI: Confidence Interval, PRP: Platelet Rich Plasma)

Table 1
Summary of the included studies

Table 1 (
continued) RCT Randomized controlled trial, FET Frozen embryo transfer, ET Embryo transfer, BMI Body mass index, HRT Hormone replacement therapy, RIF Repeated implantation failure, IVF In vitro fertilization, FSH Follicular stimulating hormone, ICSI Intracytoplasmic sperm injection, NR Not reported, PRP Platelet rich plasma

Table 2
baseline characteristics of the included studies